Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model

Diet and lifestyle produce major effects on tumour incidence, prevalence, and natural history. Moderate dietary restriction has long been recognised as a natural therapy that improves health, promotes longevity, and reduces both the incidence and growth of many tumour types. Dietary restriction differs from fasting or starvation by reducing total food and caloric intake without causing nutritional deficiencies. No prior studies have evaluated the responsiveness of malignant brain cancer to dietary restriction. We found that a moderate dietary restriction of 30-40% significantly inhibited the intracerebral growth of the CT-2A syngeneic malignant mouse astrocytoma by almost 80%. The total dietary intake for the ad libitum control group (n=9) and the dietary restriction experimental group (n=10) was about 20 and 13 Kcal x day(-1), respectively. Overall health and vitality was better in the dietary restriction-fed mice than in the ad libitum-fed mice. Tumour microvessel density (Factor VIII immunostaining) was two-fold less in the dietary restriction mice than in the ad libitum mice, whereas the tumour apoptotic index (TUNEL assay) was three-fold greater in the dietary restriction mice than in the ad libitum mice. CT-2A tumour cell-induced vascularity was also less in the dietary restriction mice than in the ad libitum mice in the in vivo Matrigel plug assay. These findings indicate that dietary restriction inhibited CT-2A growth by reducing angiogenesis and by enhancing apoptosis. Dietary restriction may shift the tumour microenvironment from a proangiogenic to an antiangiogenic state through multiple effects on the tumour cells and the tumour-associated host cells. Our data suggest that moderate dietary restriction may be an effective antiangiogenic therapy for recurrent malignant brain cancers.     

Growth and angiogenesis of human breast cancer in a nude mouse tumour model is reduced by NK4, a HGF/SF antagonist

Hepatocyte growth factor/scatter factor (HGF/SF) is a cytokine primarily produced by stromal fibroblasts and is a known angiogenic and invasion-inducing factor. It is increased in patients with breast cancer. This study examined the effect of NK4, a newly described HGF/SF antagonist, on HGF/SF-promoted growth of a human breast cancer. Both in vitro (invasion and migration assays) and in vivo (murine tumour model) methods were used to ascertain the effect of NK4 on HGF/SF from two sources: human fibroblast-derived HGF/SF and recombinant HGF/SF. In the in vitro invasion assay and migration assay, both HGF/SF and human fibroblasts, which secrete bioactive HGF/SF, increased the invasiveness and migration of the breast cancer cells (MDA MB 231). NK4 significantly reduced this invasiveness and motility. In the animal model, tumour volume and weight was significantly reduced with addition of NK4. It also suppressed HGF/SF-induced growth and markedly retarded tumour growth induced by fibroblasts (MRC5), secreting bioactive HGF/SF. Tumour angiogenesis was assessed by immunohistochemical staining of primary tissue sections using VE-cadherin (an endothelial cell specific cell-cell adhesion molecule). Again, NK4 reduced the effects of both HGF/SF and fibroblasts. We conclude that NK4 has a significant effect on the growth of human breast tumours in nude mice, particularly when stimulated by HGF/SF or fibroblasts. This may occur by decreasing angiogenesis. This gives a clear indication of the therapeutic worth of NK4.     

Macrophages promote angiogenesis in human breast tumour spheroids in vivo

An in vivo model has been established to study the role of macrophages in the initiation of angiogenesis by human breast tumour spheroids in vivo. The extent of the angiogenic response induced by T47D spheroids implanted into the dorsal skinfold chamber in nude mice was measured in vivo and compared to that induced by spheroids infiltrated with human macrophages prior to implantation. Our results indicate that the presence of macrophages in spheroids resulted in at least a three-fold upregulation in the release of vascular endothelial growth factor (VEGF) in vitro when compared with spheroids composed only of tumour cells. The angiogenic response measured around the spheroids, 3 days after in vivo implantation, was significantly greater in the spheroids infiltrated with macrophages. The number of vessels increased (macrophages vs no macrophages 34+/-1.9 vs 26+/-2.5, P<0.01), were shorter in length (macrophages vs no macrophages 116+/-4.92 vs 136+/-6.52, P<0.008) with an increased number of junctions (macrophages vs no macrophages 14+/-0.93 vs 11+/-1.25, P<0.025) all parameters indicative of new vessel formation. This is the first study to demonstrate a role for macrophages in the initiation of tumour angiogenesis in vivo.     

Green fluorescent protein imaging of tumour growth,metastasis, and angiogenesis in mouse models

We have developed a way of imaging metastases in mice by use of tumour cells expressing green fluorescent protein (GFP) that can be used to examine fresh tissue, both in situ and externally. These mice present many new possibilities for research including real-time studies of tumour progression, metastasis, and drug-response evaluations. We have now also introduced the GFP gene, cloned from bioluminescent organisms, into a series of human and rodent cancer-cell lines in vitro, which stably express GFP after transplantation to rodents with metastatic cancer. Techniques were also developed for transduction of tumours by GFP in vivo. With this fluorescent tool, single cells from tumours and metastases can be imaged. GFP-expressing tumours of the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone, and other organs have also been visualised externally by use of quantitative transcutaneous whole-body fluorescence imaging. GFP technology has also been used for real-time imaging and quantification of angiogenesis.     

The effect of tamoxifen on breast tumour vascularity

As there is experimental evidence to suggest that tamoxifen may exert an anti-angiogenic effect, the present study was designed to investigate the effect of primary tamoxifen on breast tumour angiogenesis. Fifty seven patients with large operable primary breast cancers were treated with tamoxifen (20mg daily) for between three and six months prior to definitive surgery. Clinical response to treatment was assessed by serial ultrasound measurements of tumour volume and a responding tumour was defined as one in which there was a greater than 25% reduction in volume at the end of treatment. Patients underwent a wedge biopsy at diagnosis and definitive surgery on completion of tamoxifen, thus providing tumour sections before and after treatment. Microvessel counts (mvc) were performed following staining with the endothelial cell marker, antibody to Factor VIII, and changes in mvc were correlated with response.Forty three of 57 patients had tumours that responded to tamoxifen. There was no difference in pre-treatment mvc between non-responding and responding tumours. Post-treatment mvc was significantly higher in non-responding than responding tumours. There was a significant reduction in mvc in responding tumours following treatment with tamoxifen, and a significant increase in mvc was detected in non-responding tumours. A significant correlation was demonstrated between percentage change in mvc and percentage reduction in tumour volume. This is the first study to demonstrate a reduction in breast cancer angiogenesis in tumours that have responded to primary tamoxifen in the clinical setting.     

The low-molecular-weight heparin, nadroparin, inhibits tumour angiogenesis in a rodent dorsal skinfold chamber model

Background:

Recently, low-molecular-weight heparins (LMWHs) were found to confer a survival advantage in cancer patients. The mechanism underlying this observation is unclear, but may involve inhibition of tumour angiogenesis. We aimed to examine the effects of nadroparin on tumour angiogenesis using a dorsal skinfold window chamber model in the Syrian hamster.

Methods:

AMel-3 and HAP-T1 tumours were grown in donor animals and fragments implanted in the window chambers. Animals (N=46) were treated with 200 IU of nadroparin or saline for 10 days. Repeated intravital fluorescence microscopy was performed to calculate functional microcirculatory parameters: number (N) and length (L) of microvessels, vascular area fraction (AF), and red blood cell velocity (V). Microvessel density (MVD), fractal dimension, and pericyte coverage were assessed histologically.

Results:

Active angiogenesis was observed in control animals, resulting in a significant increase in N, L, and AF. In nadroparin-treated animals, however, N and L did not increase whereas AF decreased significantly. Both groups showed an initial increase in V, but nadroparin treatment resulted in an earlier decrease in red blood cell velocity over time. Compared with control animals, nadroparin-treated animals showed a significantly lower MVD and fractal dimension but significantly higher pericyte coverage index (PCI).

Conclusions:

Taken together, these results suggest that the LMWH nadroparin inhibits tumour angiogenesis and results in microvessel normalisation.     

Tissue factor,angiogenesis and tumour progression

Tissue factor, the primary initiator of the coagulation cascade, maintains vascular integrity in response to injury. It is now recognised that, in addition to the role as a procoagulant activator, tissue factor participates in many tumour-related processes that contribute to malignant disease progression. The present review details the recent evidence supporting a role for tissue factor in tumour haemostasis, angiogenesis, metastasis and malignant cell survival. Furthermore, future research directions are discussed that may enhance our understanding of the role and regulation of this protein, which could ultimately lead to the innovative design and development of new anticancer therapies.     

The effect of hyperbaric oxygen therapy on tumour growth in a mouse model of colorectal cancer liver metastases

Background and aimsHyperbaric oxygen (HBO) therapy involves the administration of 100% oxygen at high pressure. It has been used to treat a variety of conditions including non-healing wounds, carbon monoxide poisoning, and as an adjuvant to radiotherapy or chemotherapy. The effect of HBO alone on the growth of malignancy remains controversial. This study investigates the impact of HBO on tumour growth, kinetics and microcirculation of colorectal cancer liver metastases in an experimental model.MethodsMale CBA mice were induced with colorectal liver metastases via an intrasplenic injection of a murine derived colorectal cell line. Tumours were examined using quantitative stereological analysis, histology and scanning electron microscopy of microvascular resin casts. The effect of HBO on tumour proliferation and apoptosis was quantified using immunohistochemistry.ResultsDaily exposure to HBO at 2.4 atm for 90 min had no effect on the volume of liver metastases. At day 13, HBO caused a significant reduction in tumour necrosis and proliferation compared to the non-HBO group (p = 0.002 and p = 0.008, respectively). By day 25 however, no differences were observed (p > 0.05). No differences in apoptosis or microvascular architecture were observed.ConclusionHBO did not have a tumour stimulatory effect on colorectal liver metastases and may potentially be used safely in conjunction with other therapeutic treatment modalities.     

The effect of omega-3 FAs on tumour angiogenesis and their therapeutic potential

Omega-3 fatty acid (omega-3 FA) consumption has long been associated with a lower incidence of colon, breast and prostate cancers in many human populations. Human trials have demonstrated omega-3 FA to have profound anti-inflammatory effects in those with cancer. In vitro and small animal studies have yielded a strong body of evidence establishing omega-3 FA as having anti-inflammatory, anti-apoptotic, anti-proliferative and anti-angiogenic effects. This review explores the evidence and the mechanisms by which omega-3 FA may act as angiogenesis inhibitors and identifies opportunities for original research trialling omega-3 FAs as anti-cancer agents in humans. The conclusions drawn from this review suggest that omega-3 FAs in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) found principally in oily fish have potent anti-angiogenic effects inhibiting production of many important angiogenic mediators namely; Vascular Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF), Platelet-Derived Endothelial Cell Growth Factor (PDECGF), cyclo-oxygenase 2 (COX-2), prostaglandin-E2 (PGE2), nitric oxide, Nuclear Factor Kappa Beta (NFKB), matrix metalloproteinases and beta-catenin.     

Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumour angiogenesis.

Endothelial receptor tyrosine kinases may play important roles in pathological vascular growth, particularly in tumours. In this study, immunohistochemistry was used to evaluate the expression of a novel endothelial receptor tyrosine kinase, Tie2/Tek, in the endothelium of vascular ''hotspots'' in normal breast tissue (n = 10), benign breast lesions (n = 10) and in breast tumours (n = 123). Tie2 expression was detected in the endothelium of all breast tissues examined. However, the strongest expression of Tie-2 was seen in vascular ''hot spots'' within the inflammatory infiltrate at the periphery of invasive tumours. Moreover, the proportion of Tie2-positive vessels (Tie2 counts/CD31 counts) was significantly higher in breast tumours than the proportion of Tie2-positive vessels in either normal breast tissue or benign breast lesions (P = 0.004 and 0.0001 respectively). These data are consistent with a role for Tie2 in tumour angiogenesis and demonstrate the potential use of Tie2 expression as a novel marker of the tumour vasculature.     

Use of angiogenesis inhibitors in tumour treatment

Advances in molecular biology have permitted the characterisation of mechanisms underlying angiogenesis. Angiogenesis is a crucial process in tumour pathogenesis as it sustains malignant cells with nutrients and oxygen. It is well known that tumour cells secrete various growth factors including VEGF, which triggers endothelial cells to form new capillaries. Preventing the expansion of new blood vessel networks results in reduced tumour size and metastases. Not surprisingly, numerous drugs that are currently under clinical development interfere with growth factor-derived angiogenic signals. This review aims to describe angiogenesis inhibitors and surveys their different modes of action.     

Role of genetic polymorphisms in tumour angiogenesis

Angiogenesis plays a crucial role in the development, growth and spread of solid tumours. Pro- and anti-angiogenic factors are abnormally expressed in tumours, influencing tumour angiogenesis, growth and progression. Polymorphisms in genes encoding angiogenic factors or their receptors may alter protein expression and/or activity. This article reviews the literature to determine the possible role of angiogenesis-related polymorphisms in cancer. Further research studies in this potentially crucial area of tumour biology are proposed.     

Analysis of the effects of exposure to acute hypoxia on oxidative lesions and tumour progression in a transgenic mouse breast cancer model

Background  

Tumour hypoxia is known to be a poor prognostic indicator, predictive of increased risk of metastatic disease and reduced survival. Genomic instability has been proposed as one of the potential mechanisms for hypoxic tumour progression. Both of these features are commonly found in many cancer types, but their relationship and association with tumour progression has not been examined in the same model.     

Human recombinant erythropoietin (rEpo) has no effect on tumour growth or angiogenesis

Tumour hypoxia has been shown to increase mutation rate, angiogenesis, and metastatic potential, and decrease response to conventional therapeutics. Improved tumour oxygenation should translate into increased treatment response. Exogenous recombinant erythropoietin (rEpo) has been recently shown to increase tumour oxygenation in a mammary carcinoma model. The mechanism of this action is not yet understood completely. The presence of Epo and its receptor (EpoR) have been demonstrated on several normal and neoplastic tissues, including blood vessels and various solid tumours. In addition, rEpo has been shown in two recent prospective, randomized clinical trials to negatively impact treatment outcome. In this study, we attempt to characterize the direct effects of rEpo on tumour growth and angiogenesis in two separate rodent carcinomas. The effect of rEpo on R3230 rat mammary adenocarcinomas, CT-26 mouse colon carcinomas, HCT-116 human colon carcinomas, and FaDu human head and neck tumours, all of which express EpoR, was examined. There were no differences in tumour growth or proliferation (measured by Ki-67) between placebo-treated and rEpo-treated tumours. In the mammary window chamber, vascular length density (VLD) measurements in serial images of both placebo-treated and Epo-treated rats revealed no difference in angiogenesis between the Epo-treated tumours and placebo-treated tumours at any time point. These experiments are important because they suggest that the recent clinical detriment seen with the use of Epo is not due to its tumour growth effects or angiogenesis. These studies also suggest that further preclinical studies need to examine rEpo's direct tumour effects in efforts to improve the therapeutic benefits of Epo in solid tumour patients.     

Extracellular influences on tumour angiogenesis in the aged host

Whether tumours are epithelial or non-epithelial in origin, it is generally accepted that once they reach a certain size all solid tumours are dependent upon a vascular supply to provide nutrients. Accordingly, there is great interest in how the extracellular environment enhances or inhibits vascular growth. In this minireview, we will examine key extracellular components, their changes with ageing, and discuss how these alterations may influence the subsequent development of tumour vasculature in the aged host. Because of the tight correlation between advanced age and development of prostate cancer, we will use prostate cancer as the model throughout this review.     

The effect of tumour size on ferromagnetic embolization hyperthermia in a rabbit liver tumour model.

OBJECTIVE: Ferromagnetic Embolization Hyperthermia (FEH) consists of arterially embolizing tumours with ferromagnetic particles to cause hysteretic heating upon subsequent exposure to an alternating magnetic field. The objective was to determine the effect of tumour size during FEH using a rabbit liver tumour model. METHOD: Thirty-three rabbits containing implanted hepatic VX2 carcinomas received a hepatic arterial infusion of ferromagnetic particles suspended in lipiodol. Following hysteretic heating, tumour and normal hepatic tissues were chemically analysed for iron content. Tumours were classed as small if their mass was less than the median mass for the whole group of subjects (2.1 g), and as large if their mass was greater than or equal to the median. To control for variability in tumour iron concentration, 13 small tumours were matched to 13 large tumours by iron concentration, and their heating characteristics compared. RESULTS: The heating rate in large tumours (median = 5.0 degrees C/min) was significantly greater than that in the matched small tumours (median = 2.8 degrees C/min), p = 0.006. Regression analysis determined that the slope of the heating rate vs iron concentration curve for large tumours was 1.5 times greater than that for the matched small tumours, p < 0.001. After cessation of heating in large tumours, there was continued heat dissipation into surrounding tissues, which led to anomalous temperature increases. There was an inverse linear relationship between tumour size and tumour iron concentration for a given dose of particles. CONCLUSION: For a given tumour iron concentration, larger tumours heat at a greater rate than small tumours, due to the poorer tissue cooling and better heat conduction in the necrotic regions of large tumours. This warrants further investigation as this finding could confer a significant advantage on FEH over other hyperthermic modalities in the treatment of hepatic malignancies.     

The dog mast cell tumour as a model to study the relationship between angiogenesis,mast cell density and tumour malignancy

Substantial experimental data suggest that tumour progression is associated with angiogenesis and that increase in microvessel density (MVD) is associated with increase in mast cells density (MCD). Dog mast cell tumour (MCT) is common in dog with an incidence much higher than that found in human and in both species several common biological and clinical characteristics have been demonstrated. To evaluate the role of angiogenesis in progression of this tumour and to correlate MVD and MCD, in this study a series of 78 MCT was investigated. Serial sections obtained from biopsy specimens were processed with toluidine blue staining, specific for MC identification, and by immunohistochemistry using a polyclonal antibody anti factor VIII-related antigen (FVIII-RA), used as an endothelial marker, and MVD and MCD were determined. Results showed that MVD was significantly higher in poorly differentiated (G3) MCTs than in intermediate (G2) and well differentiated (G1) MCTs and that MCD and MVD were significantly correlated in G3, but not in G1 and G2 subgroups. These data indicate that angiogenesis and MCD are significantly correlated in MCTs progression.     

Antimetastatic effect of amiloride in an animal tumour model

The diuretic Amiloride competitively inhibits the catalytic activity of the urokinase-type plasminogen activator on plasminogen in vitro. This effect was tested on a rat adenocarcinoma model and its invasive potential in the host lung. While the inhibitory effect on intact cell cultures of the tumour was slight, continuous exposure of tumour-injected animals to the drug completely prevented the formation of lung metastasis.     

Host microenvironment in breast cancer development: Inflammatory and immune cells in tumour angiogenesis and arteriogenesis

Breast cancer progression is associated with and dependent upon robust neovascularization. It is becoming clear that tumour-associated 'normal' cells, such as immune/inflammatory cells, endothelial cells and stromal cells, conspire with cancer cells in promoting this process. In particular, infiltrating immune/inflammatory cells secrete a diverse repertoire of growth factors and proteases that enable them to enhance tumour growth by stimulating angiogenesis and, as we suggest here, by promoting 'tumour arteriogenesis' – enlargement of feeding vessels supplying the expanding tumour capillary bed. Macrophages and their chemoattractants (e.g. macrophage chemoattractant protein-1) are critical for the arteriogenic process in ischaemia, and probably also in breast neoplasia. A better understanding of these various cellular and molecular constituents of breast cancer neovascularization may be useful in designing more effective therapies.